Method of culturing pancreatic islet-like tissues by a tissue complex of pancreas-derived non-endocrinal epithelial cells and vascular endothelial cells

• US 8,852,934 B2
• Filed: 03/15/2011
• Issued: 10/07/2014
• Est. Priority Date: 03/17/2010
• Status: Expired due to Fees

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Kanamune, Jun, Iwanaga, Yasuhiro, Uemoto, Shinji, Kawaguchi, Yoshiya
• Primary Examiner(s): Leith, Patricia A
• Assistant Examiner(s): Bowers, Erin M
• Application Number:
  US13/635,630
• Publication Number:
  US 20130122586A1
• Time in Patent Office: 1,302 Days
• Field of Search: None
• US Class Current: 435/366
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

According to embodiments, a method of producing insulin-producing tissues (IPTs) by culturing comprises: preparing non-endocrinal epithelial cells (NEECs) and vascular endothelial cells (VECs), which have been isolated or originated from postnatal pancreata, preferably by capturing of NEECs by collagen; culturing in vitro the NEECs and the VECs at least partly separately from each other; and then generating in vitro a tissue complex (IPTs) that contains both the NEECs and the VECs. In another embodiment, the native islet cells are seeded on a monolayer of VECs that have preferably been separately cultured and purified. In a further embodiment, a method of enriching NEECs comprises: culturing NEECs adhering to a container or substrate; removing NEECs by treating with a tissue-dissociation enzyme to leave left-over cells (LOCs) still attached on the container or substrate; and culturing NEECs in a medium conditioned by, or in the presence of the LOCs.

Claims

1. A method of producing insulin-producing tissues (IPTs) by culturing, comprising:

(a) seeding pancreatic discards, which are left-over tissues remaining after isolation of islets from a postnatal pancreas, in a first culture container or culture substrate to produce floating tissue debris and non-endocrinal epithelial cells (NEECs), wherein said NEECs adhere to the culture container or culture substrate;

(b) separating said NEECs from said floating tissue debris;

(c) culturing said separated NEECs in vitro in a first culture medium and isolating said NEECs;

(d) seeding said separated floating tissue debris into a second culture medium to produce vascular endothelial cells (VECs) and second floating tissue debris;

(e) separating said vascular endothelial cells (VECs) from said second floating tissue debris;

(f) culturing said separated VECs in vitro in a third culture medium and isolating said VECs;

(g) mixing said isolated NEECs and said isolated VECs; and

(h) generating, in vitro, a tissue complex comprising the mixture of said isolated NEECs and said isolated VECs.

2. The method according to claim 1, wherein the culture container for seeding said pancreatic discards comprises a first serum-containing culture medium and wherein said first culture container for seeding said pancreatic discards is coated with collagen or other cell-adhesion materials.

3. The method according to claim 1, wherein said culturing of said separated NEECs is performed until the emergence of clonal colonies of said separated NEECs with sizes ranging from 10-30 micrometer in diameter;

and wherein the first culture medium is a serum-free medium.

4. The method according to claim 3, wherein said serum-free medium contains keratinocyte growth factor (KGF) and, optionally, insulin-transferrin-sodium selenite supplement or other growth factor supplements.

5. The method according to claim 1, wherein the first culture medium contains serum;

and wherein step (c) further includes replacing said first culture medium during said separated NEEC culturing with a first serum-free medium containing an insulin-transferrin-sodium selenite supplement and continuing the culture; and

then replacing said first serum-free medium with a second serum-free medium.

6. The method according to claim 3, further comprising:

(1) treating said clonal colonies with a first tissue-dissociation enzyme solution upon or after said emergence to dissociate said colonies from the first culture container, filtering and/or centrifuging said clonal colonies, and removing said clonal colonies from the serum-free medium;

wherein after the removal of the clonal colonies, there are left-over cells (LOCs) that remain on the first culture container that are not removed with tissue-dissociation enzyme solution; and

(2) culturing said removed colonies in a second culture container that is coated with collagen or other cell adhesion materials in a second serum-free medium.

7. The method according to claim 6, wherein the collagen or other cell adhesion materials contain Rho-associated coiled-coil containing protein kinase (Rock) inhibitor, epidermal growth factor (EGF), and hepatocyte growth factor (HGF).

8. The method according to claim 6, wherein the second serum-free medium is supplemented with atorvastatin.

9. The method according to claim 6, further comprising culturing said LOCs in said first culture container.

10. The method according to claim 6, further comprising detaching said LOCs from said first culture container with a second tissue-dissociation enzyme solution.

11. The method according to claim 6, wherein said second serum-free medium contains proliferation-enhancing paracrine factors from said LOCs.

12. The method according to claim 1, wherein step (d) further comprises:

(d2) seeding said separated floating tissue debris into said second culture medium on a second culture container or culture substrate that is coated with collagen or other cell adhesion materials; and

(d3) expanding the VECs in the floating tissue debris subsequent to said seeding;

wherein said second culture medium contains serum.

13. The method according to claim 12, wherein step (e) further comprises:

(e2) replenishing said second culture medium with a serum-containing culture medium;

(e3) removing non-adherent cells and tissues during said replenishing; and

(e4) purifying the VECs by antibody binding to produce separated VECs.

14. The method according to claim 13, wherein said culturing of said separated VECs in vitro in a third culture medium of step (f) further comprises:

(f2) seeding said separated VECs into the third culture medium; and

(f3) replenishing said third culture medium.

15. The method according to claim 1, wherein step (g) further comprises:

(g2) treating said isolated NEECs and said isolated VECs with a tissue-dissociation enzyme solution;

(g3) centrifuging said treated NEECs and said treated VECs;

(g4) removing said centrifuged NEECs and said centrifuged VECs from said first culture medium and said third culture medium, respectively; and

(g5) placing said removed NEECs and said removed VECs into a low adherence culture container with a serum-containing differentiation culture medium.

16. The method according to claim 15, wherein step (h) further comprises:

(h2) swirling said low adherence culture container to form an initial tissue complex;

(h3) placing the low adherence culture container horizontally, without motion;

(h4) replenishing said serum-containing differentiation culture medium, thereby maturing said IPTs.

17. The method according to claim 16, wherein said serum-containing differentiation culture medium contains activin, growth factors, bone morphogenetic protein, and CaCl₂;

wherein a mixing ratio of said removed NEECs and said removed VECs is in a range from 3% to 25%, wherein said mixing ratio is obtained by dividing the cell number of the removed VECs by a sum of the cell numbers of said removed NEECs and said removed VECs; and

wherein CaCl₂ is omitted from said serum-containing differentiation culture medium upon said maturation of IPTs.

18. A method of producing insulin-producing tissues by culturing, comprising:

(a) treating a postnatal pancreas to produce natural islet cells and pancreatic discards;

(b) seeding said pancreatic discards in a first culture container or culture substrate to produce floating tissue debris and non-endocrinal epithelial cells (NEECs), wherein said NEECs adhere to the culture container or culture substrate;

(c) separating said NEECs from said floating tissue debris;

(d) seeding said floating tissue debris into a culture medium to produce vascular endothelial cells (VECs) and second floating tissue debris;

(e) separating said VECs from said second floating tissue debris;

(f) culturing said separated VECs in vitro to produce isolated VECs;

(g) culturing said isolated VECs in vitro in a manner to form a VEC monolayer;

(h) seeding and growing said natural islet cells on said VEC monolayer.

19. The method according to claim 18, wherein step (f) further comprises:

(f2) seeding and expanding said separated VECs on a second culture container or culture substrate that is coated with collagen or other cell adhesion materials to produce expanded VECs;

(f3) removing said expanded VECs from said second culture container or culture substrate; and

(f4) purifying said removed VECs by antibody binding to produce said isolated VECs.